Mystrica

colorimeter

The following pages contain details of many of the applications for the colorimeter. More details, suggestions for investigations and sample results can be viewed on the Mystrica website, www.mystrica.com

Index Page Safety Basic apparatus and Chemicals Beer’s Law Investigations with Enzymes Amylase Catechol oxidase Dopa oxidase Invertase Lactase Pepsin Phosphatase Phosphorylase Urease Measuring concentrations Chlorine Ethanol Protein Biuret test Bradford test Reducing sugars Benedict’s test DNSA reagent Vitamin C Acidified sodium thiosulphate reaction Biological membranes Buffers Citrate buffer Citric acid-sodium phosphate buffer High pH buffer Low pH buffer Phosphate buffer 32 33 34 35 36 26 28 29 30 31 24 25 21 22 5 8 9 11 13 14 16 18 20 3 3 4

2

SAFETY
The investigations described here have been developed by teachers with experience in schools and colleges worldwide. The techniques, equipment and chemicals used are, as far as possible, simple and present few hazards. Where hazards exist they are minimal, are clearly identified and the precautions for safe working are indicated. Students and teachers should • follow good laboratory practices. • complete their own risk assessments. • students should work under adequate supervision.

BASIC APPARATUS AND CHEMICALS
With a few exceptions simple laboratory apparatus is sufficient to carry out the investigations. Most schools will have access to basic equipment of various degrees of sophistication, but there is little here that could not be done in most kitchens. Other than this an accurate balance is important if solutions, buffers, etc are to be made up. A small number of the investigations benefit from the use of a bench centrifuge. Volumes can be measured with measuring cylinders or pipettes. Low cost ‘supermarket’ solutions such as using bottled water instead of distilled water and disposable plastic cups instead of laboratory glassware are perfectly acceptable. More details, suggestions for methods, investigations and sample results can be viewed on the Mystrica website, www.mystrica.com

3

Example Absorbance versus concentration of methylene blue using red.com/Experiment. There are certain conditions that must be met for the law to apply. In general colorimeters. Most substances will give good straight line responses in the range 0 ─ 0.mystrica.Beer’s Law Introduction For most applications it is preferable to measure absorbance rather than transmittance since there is a linear relationship between absorbance and the concentration of the coloured substance being measured. (see below).5 absorbance units. or at least should have be equally absorbed throughout its range of wavelengths. So precise linear relationships are not always achievable and depend on a good match between the incident waveband and the absorbed waveband. suggestions for investigations and sample results can be viewed on the Mystrica website. If this condition is not met the relationship will be non-linear. The most important of these in the context of colorimetry is that the incident light should be as near monochromatic as possible. In practice this means that there is a directly proportional straight line relationship between concentration and absorbance. www. green or blue LEDs More details. do not have the narrow wavebands that can be achieved with spectrophotometers.aspx?PageId=66 4 . Lambert-Beer or Beer-Lambert-Bouger) states that there is a logarithmic relationship between the transmission of light through a sample and the product of the absorption coefficient of the absorbing substance times the distance of the light path through the sample. Background Beer’s Law (or Beer-Lambert. though there are qualifications to be considered. whether they use LEDs or coloured filters to create their light beam.

(ptyalin). is an α-amylase. SAFETY Each student should collect and use only THEIR OWN saliva. probably using iodine solution to show the disappearance of starch when it is mixed with amylase. 5 . amylopectin. Starch is a mixture of amylose a straight chain of glucose molecules and amylose. Most starches are about 20% amylase and 80% amylopectin. or with care while wearing a dust mask. There are a number of different bacterial and fungal α-anylases available in liquid or powdered form. β-amylases digest starch by cleaving every second bond starting from one end. The product is therefore a mixture of small molecules containing two. α-amylases digest starch by randomly breaking the glycosidic bonds between glucose molecules. the most commonly used being those from mammalian saliva and pancreas. is readily available and is considered safe to use when simple precautions are followed.e. After use test-tubes and other laboratory equipment that has been in contact with saliva should be soaked in a sterilising solution such as dilute bleach before being washed up. as are fungal and bacterial amylases. Skin contact with powdered enzymes and prepared solutions should be avoided. There are many natural sources of amylase. β-amylases are obtained from plant sources. such as barley or sweet potato.Amylase Introduction The digestion of starch by amylase is likely to be one of the first enzyme reactions encountered by students. Salivary amylase. three or more glucose molecules. Background Amylase is a general term for several different enzymes that hydrolyse starch. plant amylase from sweet potatoes or from grains such as barley. If powdered enzymes from bacteria or fungi are used then precautions must be taken when preparing solutions as the enzyme powders may provoke allergic reactions. fungal amylase and bacterial amylase. (i. a mixture of maltose and dextrins). (ptyalin). amylopectin a branching chain. producing maltose. Solutions should be prepared in a fume cupboard. Suggestions for investigations • • • compare amylase from different sources measure optimum temperature and pH of amylases investigate the products of the reaction Investigations with α-amylase α-amylase is present in saliva. Salivary amylase.

Take a little water into the mouth. At 30 second intervals remove 0.mystrica.4cm3 of pH7 buffer. Reaction mixture A suitable reaction protocol for α-amylase is as follows. 0. Salivary amylase is good for showing the effects of temperature and pH on enzyme activity as it is relatively sensitive to both conditions. suggestions for investigations and sample results can be viewed on the Mystrica website.The reaction between soluble starch and α-amylase can be followed by monitoring the disappearance of the blue/black starch-iodine complex or the appearance of reducing sugars using DNSA reagent or Benedict’s reagent. to measure the maltose produced. www. Before adding it to the enzyme the substrate/buffer mixture should be equilibrated to the reaction temperature. though small ones still give good yields.1% solution of bacterial or fungal amylase).1cm3 of amylase. The extract is filtered through several layers of butter muslin. To collect salivary amylase • • Rinse the mouth out first with water. More details. (the reaction will work with water instead of buffer). (salivary amylase or a 0. The extract will keep for several weeks refrigerated without much loss of activity. or blended. 30g of sweet potato gives a useful amount of extract and we have found that large sweet potatoes give a better yield of enzyme than small. Enzyme extraction Sweet potato (Ipomoea batatas) is crushed in a pestle and mortar. • • • 0. swirl it around and spit out into a container labelled to identify the person whose saliva it contains.aspx?PageId=13 Investigations with β –amylase Sweet potato is an excellent source of β-amylase.1M HCl) Read absorbance using red light. though slower. may be easier to maintain. 35°C works well. If a centrifuge is available it can be used to remove cell debris but the extract will give good results without this stage.1cm3 of the reaction mixture and add it to 3cm3 of iodine solution. with water using 1cm3 of water for each gram of sweet potato. (2% iodine stock solution in 0.5cm3 of a 5% solution of soluble starch 4. though the optimum temperature is considerably higher than is usually given in school textbooks. Soluble starch 6 . but lower temperatures.com/Experiment. young potatoes. Enzyme activity can be shown using soluble starch as the substrate and DNSA reagent or Benedict’s reagent.

(since there are no branches in the chains to stop the progress of the enzyme).com/Experiment. Add this to 0. The buffer/substrate mixture should be allowed to equilibrate to the reaction temperature before being added to the enzyme.will not be completely broken down by β –amylase as the enzyme can only digest straight chains of glucose an is stopped by branches. At intervals of 30 seconds remove 0. suggestions for investigations and sample results can be viewed on the Mystrica website.aspx?PageId=14 Reaction mixture Suitable conditions for this reaction are a temperature of 35°C in pH6 buffer. and the blue colour obtained with iodine solution will disappear completely.1cm3 of the enzyme extract to start the reaction. An alternative method is to use amylose prepared from potato starch as the substrate as this is completely broken down to maltose.aspx?PageId=14 7 .1M HCl) Read absorbance using red light More details.com/Experiment. www. The method for preparing amylose is given on the Mystrica website. Mix 2cm3 of amylose from potato starch with 2cm3 of buffer.3cm3 DNSA reagent in a test tube. Mix 1.1cm3 of the enzyme extract to start the reaction. Add this to 0.3cm3 and add to 0. Stand the tube in boiling water for 5 minutes then add 3cm3 of cold water and read the absorbance using green light.mystrica.1cm3 into 3cm3 of iodine solution.mystrica. (2% iodine stock solution in 0.5cm3 of buffer.5cm3 of a 5% solution of soluble starch with 1. At intervals of 1 minute remove 0. Using soluble starch as the substrate and measuring the production of maltose. Using amylose as the substrate and measuring the colour of the blue complex formed between amylose and iodine. www.

SAFETY The volumes and concentrations of catechol and its inhibitors. More details. Diphenol oxidase. suggestions for investigations and sample results can be viewed on the Mystrica website. Good laboratory practices should be observed and the wearing of gloves is advised when preparing working solutions.5cm3 of catechol solution and 2. Filter the extract through several layers of butter muslin and store refrigerated.1cm3 of the enzyme extract in a cuvette and add 2. The reaction can be performed at room temperature The optimum pH is around 6.) The reaction catalysed is the oxidation of catechol to the yellow product 1. Tyrosinase. Reaction mixture For continuous reading put 0.2-benzoquinone.aspx?PageId=17 8 . Background Catechol oxidase has a number of alternative names. (Polyphenol oxidase.9cm3 of a mixture containing 0. present minimal hazards.01M and 0. We have found banana to be easiest to use.05M (final concentration in the cuvette) should give a good linear response when the concentration is plotted against the rate of reaction.mystrica. On exposure to air there is a further reaction in which the yellow benzoquinone is converted to dark brown melanin. It is easy to prepare from a number of different sources – bananas are particularly good – and the reaction is readily followed using a colorimeter.4cm3 of buffer. To prepare the enzyme blend banana with two volumes of water or just squash the banana with a fork and crush it in a pestle and mortar with two volumes of water.8 and catechol concentrations between 0. potato and apple are commonly used. and the extract retains its activity for weeks in the fridge. 20g of banana with 40cm3 of water will give plenty of extract for most purposes.com/Experiment. www. (if used). Suggestions for investigations • • • • Effect of pH on enzyme activity Effect of temperature on enzyme activity Effect of competitive and non-competitive inhibitors Enzyme kinetics Enzyme extraction Catechol oxidase can be extracted from fruits that brown on exposure of their cut surfaces to air. etc.Catechol oxidase Introduction Catechol oxidase is the enzyme responsible for the browning of fruit.

For advanced work careful preparation with measured quantities. The tissue simply needs to be crushed with cold water. L-dopa (3. Using the colourimeter detailed studies of optimum temperature. Banana and potato work well. It is not entirely clear how many enzymes and polymorphs are involved. Background Phenol oxidase enzymes that catalyse this reaction are ubiquitous in plants. It is enzymatically converted to dopaquinone by the oxidation of two hydroxyl groups followed by spontaneous conversion of dopaquinone to red dopachrome which slowly and spontaneously converts to melanin.4-dihydroxy-L-phenylalanine) is a colourless metabolite synthesised from the amino acid tyrosine. interesting. animals and fungi. (2cm3 of water per gram of tissue). etc may be desirable. reliable. centrifugation. It is simple. The advantages of this system are: • students can easily extract the enzyme for themselves • the reaction can be carried out quickly using simple methods and is wonderfully reliable and predictable • there are no safety issues • the cost is negligible • for more advanced studies the reaction can be followed by continuous colorimetry so that rates of reaction can be determined and sophisticated investigations of enzyme kinetics carried out • it has relevance to a number of important and interesting reactions and is a very good way of introducing the concept of interlinked metabolic pathways. For preference it should be filtered through muslin. addition of acid or alkali to show the effect of pH. are all relatively straightforward. pH. effects of inhibitors. effect of boiling. 9 . enzyme kinetics. etc. safe. colourful and inexpensive and can be used to introduce the concept and properties of enzymes to secondary pupils or to carry out sophisticated studies of enzyme kinetics. The enzyme system is essentially the same as catechol oxidase. the yield from potato peelings being greater than from the flesh.Dopa oxidase Introduction The conversion of L-dopa to dopachrome is a reaction that is rarely encountered in schools. Enzyme extraction The enzyme can be prepared from a variety of vegetable sources. Suggestions for investigations The system is ideal for qualitative demonstrations of enzyme action.

mystrica. Prepare solutions fresh before use and store them cold.Reaction mixture One of the reasons why this is such a good system is the ease with which the reaction can be carried out.aspx?PageId=59 10 . particularly at pH above 7.2] 0.com/Experiment.1cm3 of enzyme extract in 3cm3 of 5mM dopa will give a good reaction at room temperature. www. the maximum solubility at 25°C is 0. suggestions for investigations and sample results can be viewed on the Mystrica website. (25mM).Wt =197.5g in 100cm3. L-dopa can be dissolved in water. More details. In solution dopa will slowly decompose and darken. [L-dopa M.

Effect of pH on rate of reaction.Invertase Introduction Invertase is easy to obtain as a commercial preparation or from a culture of yeast and can be used for a variety of safe and simple yet interesting and rewarding investigations Background Invertase converts the disaccharide sucrose into the monosaccharides glucose and fructose by hydrolysing the bond between them. *Yeast extract and peptone can be replaced with 2g Marmite or Vegemite Once growth has finished and the culture has separated out the clear liquid on top should contain a high concentration of invertase. Invertase is found in the lining of the small intestine but the usual commercial source is yeast. If a centrifuge is available a short spin will remove any remaining yeast cells. We have obtained the best yield using the following recipe and growing for six hours at 35°C. Commercial preparations are available but the enzyme activity can easily be shown by culturing yeast. Experiments with immobilised enzymes Preparation of invertase Invertase can be obtained from dried yeast sold for home baking. Suggestions for investigations • • • • Effect of temperature on rate of reaction. Sucrose is a non-reducing sugar and gives negative results with Benedict’s and DNSA reagents. 11 . • Dried yeast 10g • Sucrose 5g • Yeast extract* 1g • Peptone* 1g • Water to 50cm3. These can therefore be used to monitor the enzyme by measuring the appearance of the reducing sugars glucose and fructose. Isolation and partial purification of enzyme.

www.4M sucrose solution in citrate buffer pH6 at 35°C.com/Experiment.aspx?PageId=18 12 . This method gave the results shown in the graph below using DNSA to detect reducing sugar.Reaction mixture Incubate 1cm3 of invertase solution prepared from yeast with 9cm3 of 0. After about 10 minutes the sucrose has all been converted to the reducing sugars glucose and fructose. More details.mystrica. suggestions for investigations and sample results can be viewed on the Mystrica website.

is responsible for the hydrolysis of lactose. (lactase).mystrica.1cm3 of lactase in a colourimeter cuvette and the absorbance of blue light recorded continuously. is readily available in solution. Lactase splits colourless ONPG into galactose and onitrophenol. o-nitrophenolgalactopyranoside.9cm3 of the ONPG in buffer solution was added to 0.0 and a 1% solution of a commercial liquid lactase from Aspergillus The buffering is important as the colour of the product (ONP) depends on the pH. This is not quite the same as the mammalian enzyme. Room temperature (approximately 20°C) was used throughout. (ONP). Diluted preparations do not keep well but it is easy and inexpensive to prepare fresh dilutions as required. www.aspx?PageId=20 13 . pH 7. (usually fungal). suggestions for investigations and sample results can be viewed on the Mystrica website.Lactase Introduction Lactase activity can easily be demonstrated using the artificial substrate ONPG which is converted to a yellow product by lactase. Suggestions for investigations The reaction of β-galactosidase. It is secreted by the intestinal villi in humans. The method described here uses an artificial substrate. a few drops added to milk help to make it digestible by people with lactose intolerance.1M phosphate buffer. Stored refridgerated the undiluted liquid retained over 60% of its activity after 2 years.com/Experiment. (ONPG). though the ability to produce the enzyme is lost by many people after childhood and leads to lactose intolerance. with ONPG is particularly good for investigating the effects of varying substrate and enzyme concentration on rate of reaction. an inability to digest lactose. Galactose can be used to show end-product inhibition. The colour of the product ONP is affected by pH so buffering is important and investigations of the effect of pH are not recommended. 2. More details. which is bright yellow. Background Lactase. forming glucose and galactose. Reaction mixture We have used ONPG dissolved in 0. Lactase. being a β-galactosidase. Continuous colorimetry can be used to obtain graphs of reaction rates.

dense suspension.Pepsin Introduction The slow breakdown of albumen proteins gives good results with continuous colorimetry and is well suited for the study of the effect of pH on enzyme activity. Preparation of an egg white suspension • • • • • separate the white from the yolk – depending on the size of the egg this will probably be about 30cm3 add 9 volumes of water – 270cm3 to a 30cm3 egg (this seems to work well with a fresh egg. for 5-10 minutes. a larger molecule that is activated by hydrochloric acid to the smaller pepsin molecule. Each new temperature should have its pH individually adjusted using a pH meter. with gentle stirring. but older eggs may need less water to achieve a good. Suggestions for investigations It is particularly suitable for demonstrating the effect of pH. The effect of temperature on enzyme activity can also be investigated.8) Pepsin does not break the bonds between every amino acid so the products of the reaction are polypeptides of varying lengths. Pepsin can only work in a low pH environment. though this cannot be done with continuous colorimetry because of difficulties maintaining temperatures very different from ambient in the colorimeter. This can be diluted 1:1 with water or buffer for use. You should be left with an opaque suspension. (do not boil this – the suspension will form at around 70°C) pour the mixture through 2-4 layers of butter muslin cloth to remove large particles.) stir gently to disperse the egg white stand the mixture in a bath of hot water. The results may be affected by changes in pH with changing temperature. which will slowly clear as the albumen protein is broken down into small. (optimum pH~1. 14 . It is produced as pepsinogen. Pepsin’s low optimum pH is noteworthy and interesting comparisons can be made with other enzymes which usually have much higher pH optima. Pepsin can conveniently be assayed using a cloudy suspension of egg white (albumen). soluble polypeptides. Background Pepsin is a proteolyic (protein splitting) enzyme produced in the gastric glands lining the stomach of vertebrates.

www.aspx?PageId=21 15 . suggestions for investigations and sample results can be viewed on the Mystrica website.com/Experiment.1 at 25°C + 0.mystrica. More details. The absorbance was measured using blue light.Reaction mixture The results illustrated below were obtained with 2.2cm3 of 5% pepsin solution.8cm3 egg white suspension adjusted to pH2.

Do not use a buffer containing phosphate.1M Phosphate buffer pH6 (cm3) 0 0. The assay method described here is for an acid phosphatase that can be obtained from germinated seeds. (beansprouts).5 0. Suggestions for investigations End product inhibition: The action of phosphatase releases phosphate which has a powerful inhibitory effect on the reaction. This is easily demonstrated by adding small amounts of phosphate buffer at the same pH to the citric acid-sodium citrate buffer. though other sources could be investigated. a phosphatase with an optimum pH below 7) is readily obtained from germinated mung beans. This reaction is particularly suitable for studying the effects of end product inhibition since the addition of small amounts of phosphate has a marked effect on the rate of the reaction. For example: 0. Background An acid phosphatase (i.5 Enzyme solution (cm3) 0. 16 .8 5.7 Enzyme extraction To prepare an extract of acid phosphatase crush 20g of germinated mung beans.4 2. in a pestle and mortar and add 10cm3 of water. Phenolphthalein is pink in alkaline solution and the absorbance can be measured using the colorimeter and green light.Phosphatase Introduction A phosphatase is an enzyme that releases a phosphate group from its substrate by hydrolysis.5 [Phosphate] mM 0 2.1M Citric acid-Sodium citrate buffer pH6 (cm3) 2. To measure the activity of the enzyme a good substrate is phenolphthalein phosphate which is colourless until it is hydrolysed to phenolphthalein.e. Adding the reaction mixture to an alkali also serves to stop the reaction.3 0.) Filter the mixture through several layers of muslin and store refrigerated. (Alternatively add 10cm3 of citric acid-sodium citrate buffer pH6.2 1% phenolphptalien diphosphate (cm3) 0.5 0.1 0. (beansprouts).5 2.5 0.5 0.

5cm3 1% phenolphthalein diphosphate tetrasodium salt in water 0.5cm3 citric acid-sodium citrate buffer pH6 0.5cm3 of the reaction mixture to 2.mystrica. timing and temperature. More details.5cm3 1% 0.Reaction mixture The following method works well but there are many potential modifications of the volumes. Read absorbance using green light. suggestions for investigations and sample results can be viewed on the Mystrica website.5cm3 10% sodium carbonate solution.com/Experiment.5cm3 enzyme preparation Incubate for 10 minutes at 25°C then add 0. www. The end point should turn pink on addition to sodium carbonate. • • • 2.aspx?PageId=22 17 .

The direction of the reaction depends on the ratio of glucose-1-phosphate : inorganic phosphate. The reaction requires priming with small fragments of partially hydrolysed starch which can grow with the addition of further glucose molecules. The resulting liquid can be used as it is or centrifuged to remove suspended particles.Phosphorylase Introduction Potato phosphorylase can be used to demonstrate the synthesis of starch. Background Phosphorylase. It also requires the absence of inorganic phosphate since this will cause the reaction to proceed in the opposite direction. Extraction of potato phosphorylase Try to keep everything as cold as possible during this extraction. the reaction proceeds in the opposite direction and starch is synthesised by the addition of successive glucose molecules to pre-existing polysaccharide chains. It can also be used to investigate the equilibrium in a reversible reaction.1M citric acid-sodium citrate buffer pH6 in the ratio 2g potato : 1cm3 buffer. However. if provided with glucose-1-phosphate and in the absence of inorganic phosphate. By adding phosphate during the course of the reaction the direction can be reversed and synthesis of starch becomes degradation of starch. Suggestions for investigations Starch phosphorylase extracted from potato can be used to demonstrate the synthesis reaction in the presence of glucose-1-phosphate. In both of these investigations the reaction can be followed using a colorimeter to measure the starch-iodine complex. Use a blender. Filter the liquid through four layers of muslin and allow the filtrate to stand to let residual starch settle. is usually associated with the breakdown of starch or glygogen by the release of terminal glucose molecules as glucose-1-phosphate. This system can also be used as a good demonstration of reversibility and equilibrium in an enzyme reaction. 18 . or a pestle and mortar to liquidise small pieces of potato with 0. Alternatively the liquid can be filtered through filter paper.

Using a phosphate-free buffer the reaction can be run in the direction of starch synthesis. synthesis or degradation of starch).1M HCl. www. The reaction can be carried out at room temperature. the results reported here were all obtained at 25°C.1cm3 primer 0.5cm3 enzyme 0. then by adding a phosphate buffer it can be reversed and the starch begins to be degraded.1M citric acid-sodium citrate buffer pH6 and 0. A satisfactory primer can easily be prepared by acid hydrolysis of soluble starch. The direction of the reaction.Primer The synthesis of starch by potato phosphorylase can only proceed if the reaction mix is primed by the addition of small fragments of starch at least four glucose molecules in length. Comparing this with the effect of the addition of a phosphate-free buffer acts as a control. • Add 0. • Stand both test tubes in boiling water for a few minutes to heat up then mix the contents and let the mixture stand in boiling water for 4 minutes.4cm3 of 0.mystrica. The buffers used were 0.1M. 0.1M phosphate buffer pH6. We have found the following method gives good results. pH6). The equilibrium point occurs at a phosphate:G-1-P ratio of about 7:1. suggestions for investigations and sample results can be viewed on the Mystrica website.5cm3 samples were taken and added to 3cm3 of iodine solution containing 0. (i.1cm3 of this primer to 5cm3 of the reaction mixture Reaction mixture We have used the reaction mixture given here successfully.01M glucose-1-phosphate in citric acid-citrate buffer.e. More details. • In a second test tube put 1cm3 of 2M hydrochloric acid. • Remove the test tube and add 1cm3 of 2M sodium hydroxide to stop the reactions and neutralise the pH.com/Experiment. showing that in this case the reaction will continue to run in the direction of the synthesis of starch. • Place 2cm3 of a 10% solution of soluble starch in a test tube. • • • • 4.aspx?PageId=23 19 . (0. depends on the ratio of glucose-1-phosphate to inorganic phosphate.

Background Urease from the jack bean (Canavalia ensiformis) was the first enzyme to be crystalised.6.0. www.mystrica.2cm3 of an enzyme solution made by adding one urease tablet to 80cm3 of water. It breaks down urea into carbon dioxide and ammonia.01% bromothymol blue and 0. The results shown (absorbance versus time) were obtained at 25°C using 3cm3 of 10mM urea containing 0. for which discovery James Sumner was awarded a Nobel Prize for Chemistry in 1946.aspx?PageId=64 20 . The reaction can be followed by measuring the change in absorbance of red light with the colorimeter Reaction mixture We have obtained good results using a 0.01% w/v solution of bromothymol blue adjusted so that it is just yellow. A number of tests for urease utilise the fact that the ammonia released causes a rise in pH which can be detected with suitable indicators. The method described here uses bromothymol blue indicator which changes from yellow to blue as ammonia is released during the reaction. The method described here gives good results with continuous colorimetry using bromothymol blue indicator which undergoes a change from yellow to blue as the pH rises from 6 to 7. Jack bean urease is commercially available in powder or tablet form .the enzyme should be freshly prepared as it does not seem to keep well.Urease Introduction There are a number of different ways of measuring urease activity. More details. suggestions for investigations and sample results can be viewed on the Mystrica website. pH about 6.com/Experiment.

2cm3 of this to 80 cm3 distilled water. either in swimming pools or in the treatment of drinking water. Read the absorbance with green light.2cm3 of a 2% solution of soluble starch in 0.5cm3 ethanoic acid will give a chlorine concentration of 8ppm (8mg/l). (See Sample results on the toolbar) More details. These are the free active chlorine compounds in the water as opposed to the bound chlorine that has reacted with other compounds such as ammonia.Chlorine Introduction The method described here can be used to determine the free chlorine content of unknown solutions such as swimming pool water and is sensitive enough to measure the chlorine content of most domestic tap water Background When water is treated by chlorination. initially a 1% dilution so that a reasonable volume can be measured in preparing the final dilution.aspx?PageId=69 21 . This volume (cm3) in 100cm3 of distilled water acidified with 0. Depending on the pH there are also varying amounts of chlorine (Cl2) and hypochlorite ions (ClO-). For other sources of chlorine divide 0. Cl2 + 2KI → I2 + 2KCl The intensity of the colour produced can be measured using the colorimeter (blue light) and compared with standards of known concentration to measure free chlorine.084 by the percentage concentration of sodium hypochlorite.) Test method Add 2cm3 of the sample to be tested to 0. (This is best done in two steps. suggestions for investigations and sample results can be viewed on the Mystrica website.1M potassium iodide solution. This was acidified by the addition of 0. The free chlorine compounds react with potassium iodide to release iodine. Preparing standard solutions of known chlorine concentration Standard solutions can be prepared from household chlorine bleaches which typically contain 3-6% sodium hypochlorite (NaClO). We used ‘Milton’ sterilising fluid which is 2% sodium hypochlorite.com/Experiment.mystrica. To prepare a solution containing 8ppm (8mg/l) chlorine we diluted the Milton 1/100 then added 4.5cm3 of ethanoic acid and made up to a final volume of 100cm3. www. it is present mainly as hypochlorous acid (HOCl). The sensitivity of this method is increased by adding soluble starch solution which reacts with the iodine released giving an intense blue colour that can be measured using the green or red light on the colorimeter.

The method works without the addition of NaOH though the green colour is less apparent. 3CH3CH2OH + K2Cr2O7 + 4H2SO4 → 3CH3CHO + Cr2(SO4)3 + K2SO4 + 7H2O The acetaldehyde is itself oxidised to acetic acid. The method we have used and described in the example illustrated used 0.5cm3 of sample to which was added 1cm3 of 1M H2SO4. Incubation was at 50°C for 30 minutes. 3CH3CHO + K2Cr2O7 + 4H2SO4 → 3CH3COOH + Cr2(SO4)3 + K2SO4 + 4H2O As the dichromate oxidises the reactants it is reduced to trivalent chromium Cr3+ which is green. though the only example we have found where this is significant is the oxidation of glucose. 22 . It should be noted that other organic molecules can also be oxidised by dichromate with similar results. The intensity of the green colour can be measured with the colorimeter and compared to known concentrations to measure the concentration of ethanol in the original sample. Different concentration of the acid and different incubation temperatures can be tried and will give different results.Ethanol Background Under acidic conditions ethanol is oxidised to acetaldehyde by potassium dichromate. Method The test sample is acidified with H2SO4 and heated. age 14) and should be labelled 'Corrosive'. The colour change can be measured at this point or after the addition of 2cm3 of 2M NaOH (corrosive) to remove the remaining dichromate and reveal the green chromium Cr3+ SAFETY 1M H2SO4 should be labelled 'Irritant' 2M NaOH should not be used with pupils before Yr9 (S3.

aspx?PageId=68 23 .46%). e. less powerful reagents can be used to reduce the safety issues.com/Experiment. www.mystrica. (o.g to measure the ethanol in of alcoholic drinks. For higher concentrations of ethanol. suggestions for investigations and sample results can be viewed on the Mystrica website. More details.Standard curve Using the method described above the results shown below were obtained.1M = 0.

com/Experiment. If a precipitate forms 0.BIURET TEST Qualitative or quantitative test for protein Background Biuret reagent reacts with peptide bonds.75M sodium hydroxide.75g 0. www. turning from blue to purple. Unlike the Bradford test it will give equally good results with any protein.1-10 mg/cm3. SAFETY Biuret reagent contains 0.5H2O Sodium potassium tartrate Dissolve in 250cm3 H2O Solution 2 Sodium hydroxide Dissolve in 150cm3 H2O 15g 0. Allow the mixture to stand for 10 minutes then read absorbance in green light. If the solution turns purple it means that protein is present.5g potassium iodide can be added Method • • To perform the quantitative test add 2 cm3 of Biuret reagent to 0.mystrica.aspx?PageId=27 24 . but it is unable to detect the low concentrations that can be achieved with the Bradford test A qualitative test can be performed simply by adding equal quantities of 1% sodium (or potassium) hydroxide and a 1% solution of copper sulphate to the sample.3g Add 2 to 1 mixing thoroughly and make up to 500cm3. More details and sample results can be viewed on the Mystrica website. The method described here uses quantitative Biuret solution which will give a good estimate of protein concentrations in the range 0. THE USE OF EYE PROTECTION MUST BE STRICTLY ENFORCED In the event of eye contact flood the eye gently with running water for ten minutes and seek medical attention Solution 1 Copper sulphate.5 cm3 of sample.

We have used powdered egg white from the home-baking section of our local supermarket as the standard with results comparable to BSA. casein and gelatin all give different responses. The reagent contains Coomassie Blue dye which is light brown in the reagent but blue when bound to protein. (stock solution diluted 1+4 with water). longer intervals will not affect the result.5cm3 of the reagent.BRADFORD TEST Quantitative test for protein Background The Bradford assay is a very good. which is partially hydrolysed collagen. contains very few of the amino acids to which the reagent is sensitive. principally arginine. This is relatively expensive.25cm3 of the sample solution. Gelatin has a very weak response to Bradford reagent since the protein. The dye will stain skin and clothes.mystrica. Stock solution • • • • Dissolve 50mg Coomassie Blue in 20cm3 methanol Add this to 60cm3 phosphoric acid Make up to 100cm3 with water Label the stock solution 'CORROSIVE' Method Add 2. More details and sample results can be viewed on the Mystrica website. Allow the mixture to stand for ten minutes then read the absorbance using red light. method of detecting microgram quantities of protein. For example albumin. www. and simple. Microassay: The sensitivity can be increased by adding 0. The standard usually employed is bovine serum albumin.com/Experiment.5cm3 of undiluted stock solution to 2cm3 of the solution to be tested. to 0. However the test is specific for certain amino acids.aspx?PageId=26 25 . SAFETY The stock solution contains phosphoric acid (50%). so not all proteins give the same reaction. Diluted for use the reagent is irritant. Ten minutes allows full development of the colour. The method will detect concentrations down to about 5 g per cm3. (BSA).

001M. Add 2 to 1 with rapid stirring Add 0.7g Dissolve in 50mls H2O. Quantitative Benedicts reagent Solution 1 Sodium citrate 100g Sodium carbonate (anhydrous) 32. so this test can be used semi-quantitatively to indicate approximate concentrations. SAFETY WEAR EYE PROTECTION TAKE CARE WITH BOILING WATER Benedicts reagent Solution 1 Sodium citrate 86. The resulting colour change depends on the type and concentration of sugar. Using a colorimeter you can obtain accurate. Add 2 to 1 with rapid stirring then dilute to 500mls Positive result on boiling with reducing sugars The stock solution does not require a hazard warning label. fully quantitative determinations of concentration down to 0. Lower concentrations can be detected rather more easily and in smaller volumes using DNSA reagent.5H2O 9g Dissolve in 50mls H2O. The stock solution does not require a hazard warning label. (180 g of glucose/cm3).5g Potassium thiocyanate 62.BENEDICT’S TEST Qualitative or quantitative test for reducing sugars Background Benedict’s solution reacts with reducing sugars on heating and reduces the Cu(II) ion to Cu(I) producing a precipitate of red copper oxide.5H2O 8.13g potassium hexacyanoferrate (II) then dilute to 500mls For colourimetric use dilute 35mls of this solution to 100mls with water. An alternative version of Benedicts reagent for quantitative testing (QBS) contains potassium thyocyanate and does not form red copper oxide. 26 .5g Sodium carbonate (anhydrous) 50g Dissolve in 400mls H2O Solution 2 Copper sulphate.5g Dissolve in 400mls H2O Solution 2 Copper sulphate. This is about 5 times lower than the concentrations detectable with test strips. Instead the presence of reducing sugar is measured by the loss of the blue colour of copper sulphate and a white precipitate is formed which will settle out or can be removed by filtration before measuring the colour of the filrate.

com/Experiment.aspx?PageId=19 27 . A colour change through green to yellow. Quantitative • • • • Add 2cm3 of QBS to 4cm3 of sample in a test tube. Stand the test tube in boiling water for a few minutes.mystrica. www. Stand the test tube in boiling water for 5 minutes Allow the tubes to stand until the precipitate settles. Measure the absorbance using red light More details and sample results can be viewed on the Mystrica website. brown and finally to red indicates the presence of reducing sugar. or filter to remove the precipitate.Methods Qualitative • • • Add about 5cm3 of the reagent to a small amount of sample in a test tube.

then diluted with water and the colour read using a colorimeter.5 dinitrosalycylic acid (DNSA) reagent changes from yellow to red. There is no need to filter after boiling and small volumes of test solutions can be used. SAFETY WEAR EYE PROTECTION TAKE CARE WITH BOILING WATER DNSA reagent contains 0.3cm3 of the sample to be tested to 0. label the stock solution 'IRRITANT' Method • • • Add 0. Small volumes of the reagent and test sample are boiled for 510 minutes. dilute to a final volume of 100cm3 with water. www. Add 3cm3 of water and read absorbance with green light (525nm).mystrica.DNSA REAGENT Quantitative test for reducing sugars Background On boiling with reducing sugars 3.4M NaOH To prepare 100cm3 of reagent • • • • • dissolve 1g of 3.com/Experiment. (90 g of glucose/cm3).aspx?PageId=16 28 . Concentrations of reducing sugar down to below 0. (KNaC4H4O6 4H2O) add 20cm3 of 2N NaOH.5mM. Stand the test tube in boiling water for 5 -10 minutes. More details and sample results can be viewed on the Mystrica website.3cm3 of DNSA reagent in a test tube.5-dinitrosalicylic acid in 50cm3 of water. can be detected using this test. (typically 0.3cm3 for a standard cuvette). slowly add 30g sodium potassium tartrate tetrahydrate.

Make up to 100cm3 with water and store in a glass bottle. The Tolerable Upper Intake level in USA is 2000mglday for a 25 year old male. Background Vitamin C. though its history goes back to ancient folk knowledge of the need for fresh plant material and raw animal flesh to prevent disease. cheaper and easier than the alternative. and 60-95mglday in USA depending on age and sex. The RDA (Recommended Dietary Allowance) for vitamin C is 40mgjday (UK). was the first vitamin to be artificially synthesised in 1935. bones.com/Experiment. Method The methods for detecting vitamin C generally make use of its anti-oxidant property. • Add O.5cm3 of test solution to 3cm3 of the starch-iodine solution.To prepare 100cm3 of stock solution put 1g of potassium iodide into a mortar and dissolve this in the smallest amount of water possible. A method often used in schools is the reduction of DCPIP from blue to colourless. fruits and vegetables is an easy practical and gives good quantitative results. though independent sources have recommended daily intakes higher than this. 45mglday (WHO). Iodine solution will not keep well after the stock solution has been diluted or in plastic containers. immobilisation and eventual death. 29 . lack of the vitamin eventually leads to the symptoms associated with scurvy resulting from collagen failure. For a standard curve a range of concentrations between 0 and 0.Vitamin C Introduction The determination of the vitamin C content of different drinks. skin. accuracy and precision.2g of soluble starch in about 50cm3 of boiling water. • To prepare a stock solution of starch-iodine dissolve O.mystrica. failure of wounds to heal. ligaments and elsewhere. Add 1g of iodine and grind to a smooth mixture.aspx?Pageld=28 * Iodine stock solution . • For colorimetry this should be diluted 1:4 with water to give an absorbance between 1 and 1. The most important role of vitamin C is in the formation of collagen. Early symptoms include spongy bleeding gums. www.1g/l are suitable. • Add 2cm3 of stock iodine solution* and make the final volume up to 100cm3 with water. (ascorbic acid). liver spots on the skin and lethargy followed in the later stages by loss of teeth. The method recommended here is the decolourisation of the blue/black starch-iodine complex which we have found as accurate. suggestions for investigations and sample results can be viewed on the Mystrica website. More details. Using known concentrations of ascorbic acid to produce standard curves allows accurate determination of concentration and opportunities for students to practice techniques such as the preparation of dilution series.5 using red light. cartilage. the ubiquitous structural protein component found in connective tissues. The practical generates results that students find interesting and relevant.

com/Experiment. Na2S2O3 + 2HCl → SO2 + S + H2O + 2NaCl The colorimeter can be used to measure the rate of this reaction since the increasing opacity scatters the light passing through. Sodium thiosulphate reacts with acid to produce sulphur which is deposited in the solution causing it to become opaque. www.mystrica. More details.REACTION OF SODIUM THIOSULPHATE WITH ACID Introduction This is a well-known reaction found in most chemistry books and often carried out at GCSE level. suggestions for investigations and sample results can be viewed on the Mystrica website.aspx?PageId=67 30 .

The red pigment of beetroot is retained in the cells and only escapes into the surrounding medium if the membrane is damaged. Cylinders about 10mm long with a diameter of about 5mm are ideal. www. (plasma membrane).aspx?PageId=44 31 . The amount of pigment leaking through the membrane into the surrounding liquid can be observed qualitatively by eye or measured accurately using the colorimeter.mystrica.Biological membranes Introduction Use beetroot to show how membrane integrity is affected by treatments that damage either the protein or the phospholipid component of the membrane.com/Experiment. is composed primarily of a bilayer of phospholipid molecules with a mosaic of protein molecules embedded in and attached to it. Both these components are necessary for the membrane to fulfil its role of maintaining the integrity of the cell contents while allowing selected substances to cross the membrane. (If you do not have a cork borer just cut pieces about this size with a knife.) The pieces must be thoroughly washed in running water to remove all surface pigment released from damaged cells. More details. The effect of treatments that damage either the phospholipids or the protein components can be investigated using beetroot. Method Use small pieces of beetroot cut using a cork borer. suggestions for investigations and sample results can be viewed on the Mystrica website. Background The cell membrane.

66 0.1M solution of trisodium citrate dihydrate.Buffers acid0.5 59 54 49.2 3.1M trisodium citrate (cm3) Volume of 0.8 5.21 1.5 41 46 50.0 3.5 74.6 4.19 2.41g/l).2 5.76 1.34 0.32 2.07 1.6 3.5 63.1M solution (cm3) 3.84 0.4 3.5 21 16 11.53 1.2 82 77.5 73 68.04 2. C6H5 O7Na3 2H2O (29.1M citric acid (cm3) 0. Or dissolve the masses shown and make up to 100cm3 with water pH 0.0 5.72 1.17 Volume of 0.93 0.91 2.5 25.44 0.04 0.4 5.8 4.01g/l) and a 0.53 0.5 60 65 69.24 1.47 2.63 1. C6H8 O7 H2O (21.60 2.1M solution of citric acid monohydrate.5 36.5 27 31.0 4.2 Prepare a 0.5 79 84 88.1M solution (cm3) 18 22.1M Citric acid-Sodium citrate buffer buffer – pH range 3.0 – 6.64 0.79 0. Mix the volumes shown in the table.5 44.54 0.63 1.71 32 .24 0.49 1.33 1.4 4.2 4.93 1.5 8 Mass in 100cm3 (g) 1.13 1.8 6.35 1.74 0.5 92 Mass in 100cm3 (g) 0.44 1.6 5.0 6.5 40 35 30.5 55.

8 7.8 5.15 66.16 3.69 3.37 0.97 0.75 17.75 77.55 41.15 79.5 38.99 4.6 5.0 4.54 2.04 2.51 6.7 48.33 4.2 6.23 6.6 55.21 5.6 Volume of 0.8 4.29 1.77 1.1M solution (cm3) 89.0 6.5 32.13 Na2HPO4 12H2O Volume of 0.25 82. Or dissolve the masses shown and make up to 100cm3 with water Citric acid pH at 25°C 2.77 2.25 42.55 36.84 3.2 3.2 4.48 0.55 24.45 75.6 –7.96 5.4 44.8 3.65 13.31 2.4 44.25 50.42 1.85 33.25 72.16 4.4 4.02 0.6 2.47 1.7 28.90 6.15 6.65 0.53 3.1 69.25 22.64g/l) Mix the volumes shown in the table.85 20.27 0.6 55.9 30.97 3.52 4.01g/l) and a 0.9 53. C6H8 O7 H2O (21.35 86.4 6.0 60.07 1.2 5.4 3.2 35.1 46.93 0.45 63.8 6.35 3.4 7.58 1.12 1.77 0.1 84.75 58.53 5.0 3.78 1.2M solution of Na2HPO4 12H2O (71.23 1.5 46.74 4.87 1.75 49.2 7.83 0.6 6.0 7.71 33 .6 4.50 1.14 1.9 15.3 51.5 67.2M solution (cm3) 10.4 5.71 0.5 61.45 58.1M solution of citric acid monohydrate.0 39.76 2.6 Prepare a 0.05 9.75 27.8 64.65 Mass in 100cm3 (g) 0.17 1.67 1.35 Mass in 100cm3 (g) 1.3 71.95 90.88 0.0 5.acidphosphate Citric acid-Sodium phosphate buffer – pH range 2.57 0.5 53.36 1.85 93.6 3.19 0.

62g/l) and sodium hydrogen carbonate (NaHCO3) (8.9 10.5 9.6 –7.1M solutions of sodium carbonate (Na2CO3 10H2O) (28. 39.43 1. 245 (1945) 34 .High pH buffer – pH range 2.6 Prepare 0.08 Delroy & King.3 10.1M solution (cm3) 10 20 30 40 50 60 70 80 90 Mass in 100cm3 (g) 0.50 0.4 9.17 0.00 2.2 9.67 0.8 9.8 at 37°C 8.1 9.6 Na2CO3 Volume of 0.9 10.1 10.57 0. Or dissolve the masses shown and make up to 100cm3 with water pH at 20°C 9.58 NaHCO3 Volume of 0.7 9.3 10.5 9.5 10.29 2.76 0.72 2.42 0.25 0.1 10.8 9.4 9.86 1.34 0.2M solution (cm3) 90 80 70 60 50 40 30 20 10 Mass in 100cm3 (g) 0.4g/l) Mix these in the volumes shown in the table. J.59 0.29 0.14 1. Biochem.

0 10.1 1.9 Bower & Bates.6 26.7 16. Bur.5 33.2M HCl added (cm3) 67.4 1. Res. natn.5 5.8 1.2 13. 55.9 2.92g/l).2 1.1 3.2M solution of potassium chloride (KCl) (14.Low pH buffer – pH range 1.6 1. To 25cm3 of this solution add the volume of 0.2 Prepare a 0.0 –2. Stand. 197 (1955) 35 .0 1.3 1.6 20.1 6. pH at 25°C 1.0 2. J.2 Volume of 0.0 52.7 1.2 8.5 1.1 2.2M HCl shown in the table and make up to 100cm3 with water.8 42.

0 Prepare 0.65 Mass in 100cm3 (g) 1.2 6.4 7.44 0.64g/l) and NaH2PO4 2H2O (31.5 4.37 1.1M Phosphate buffer – pH range 5. after Sørensen.0 43.20 0.2M solution in 100cm3 (cm3) 46.75 24.5 30.15 9.44 0.0 7.8 –8.58 2.0 6.4 6.5 45.61 0.34 1.5 6.44 1.85 40.0.08 5.5 19.2M solution in 100cm3 (cm3) 4.0 Gomori.2M solutions of Na2HPO4 12H2O (71.80 0.6 6.75 47.25 18.8 6.75 36.90 3.25 2.5 14.6 7.29 0.28 3.0 6. Methods in Enzymology 1.0 9.76 2.2 7.21g/l) Mix the volumes shown in the table and make the total volume up to 100cm3 or dissolve the masses indicated in water and make up to 100cm3 Na2HPO4 12H2O pH at 25°C Volume of 0.15 0.25 25.95 1.66 0.27 1.30 0.75 31. 143 (1955) 36 .8 7.0 40.39 NaH2PO4 2H2O Volume of 0.5 36.12 3.25 13.98 0.13 0.5 43.19 2.35 Mass in 100cm3 (g) 0.8 8.

Sign up to vote on this title
UsefulNot useful